Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors

Vermeíre, Jolien; Naessens, Evelien; Vanderstraeten, Hanne; Landi, Alessia; Iannucci, Veronica; Nuffel, Anouk Van; Taghon, Tom; Pizzato, Massimo; Verhasselt, Bruno

doi:10.1371/journal.pone.0050859

Cited by 195 publications

(177 citation statements)

References 52 publications

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“…Reverse transcriptase (RT) activity in the supernatants of 293T cells transfected with pBru3-based constructs carrying the luciferase gene in place of the nef gene in the absence of the VSV-G envelope to prevent infection of cells with newly produced virions was determined using a SYBR green-based quantitative PCR assay (64). Transfection efficiency was assessed by measuring the luciferase activity of cells lysed at 48 h after transfection with the Luciferase Cell Culture Lysis 5ϫ reagent and luciferase assay system (Promega).…”

Section: Methodsmentioning

confidence: 99%

Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

Saito

Henning

Serrao

et al. 2016

J Virol

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Cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a host factor that interacts with the HIV-1 capsid (CA) protein, is implicated in diverse functions during the early part of the HIV-1 life cycle, including uncoating, nuclear entry, and integration targeting. Preservation of CA binding to CPSF6 in vivo suggests that this interaction is fine-tuned for efficient HIV-1 replication in physiologically relevant settings. Nevertheless, this possibility has not been formally examined. To assess the requirement for optimal CPSF6-CA binding during infection of primary cells and in vivo, we utilized a novel CA mutation, A77V, that significantly reduced CA binding to CPSF6. The A77V mutation rendered HIV-1 largely independent from TNPO3, NUP358, and NUP153 for infection and altered the integration site preference of HIV-1 without any discernible effects during the late steps of the virus life cycle. Surprisingly, the A77V mutant virus maintained the ability to replicate in monocyte-derived macrophages, primary CD4 ؉ T cells, and humanized mice at a level comparable to that for the wild-type (WT) virus. Nonetheless, revertant viruses that restored the WT CA sequence and hence CA binding to CPSF6 emerged in three out of four A77V-infected animals. These results suggest that the optimal interaction of CA with CPSF6, though not absolutely essential for HIV-1 replication in physiologically relevant settings, confers a significant fitness advantage to the virus and thus is strictly conserved among naturally circulating HIV-1 strains. IMPORTANCECPSF6 interacts with the HIV-1 capsid (CA) protein and has been implicated in nuclear entry and integration targeting. Preservation of CPSF6-CA binding across various HIV-1 strains suggested that the optimal interaction between CA and CPSF6 is critical during HIV-1 replication in vivo. Here, we identified a novel HIV-1 capsid mutant that reduces binding to CPSF6, is largely independent from the known cofactors for nuclear entry, and alters integration site preference. Despite these changes, virus carrying this mutation replicated in humanized mice at levels indistinguishable from those of the wild-type virus. However, in the majority of the animals, the mutant virus reverted back to the wild-type sequence, hence restoring the wild-type level of CA-CPSF6 interactions. These results suggest that optimal binding of CA to CPSF6 is not absolutely essential for HIV-1 replication in vivo but provides a fitness advantage that leads to the widespread usage of CPSF6 by HIV-1 in vivo.T he HIV-1 capsid (CA) protein forms the protein core that envelopes viral enzymes and genetic material following fusion with the plasma membrane (1-3). Uncoating of the viral core is a fine-tuned process that regulates downstream events, including reverse transcription, nuclear entry, and integration targeting (4-9). It is likely that HIV-1 exploits multiple host factors that can directly interact with the viral CA protein for proper uncoating in virus-infected cells. These host factors likely in...

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Section: Methodsmentioning

confidence: 99%

Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

Saito

Henning

Serrao

et al. 2016

J Virol

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“…On day 10, the thymocyte number per well was determined using 10-m latex beads (Coulter), and the cells were analyzed by flow cytometry or stored as pellets at Ϫ80°C. Viral production in supernatants was quantified at day 10 postinfection of CD11c neg CD14 neg CD123 neg T-cell receptor ␥␦-negative (TCR␥␦ neg ) thymocytes by measuring RT activity using SYBR green product-enhanced RT (SG-PERT), as described previously (29,30).…”

Section: Methodsmentioning

confidence: 99%

Thymic HIV-2 Infection Uncovers Posttranscriptional Control of Viral Replication in Human Thymocytes

Nunes‐Cabaço

Matoso

Foxall

et al. 2015

J Virol

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A unique HIV-host equilibrium exists in untreated HIV-2-infected individuals. This equilibrium is characterized by low to undetectable levels of viremia throughout the disease course, despite the establishment of disseminated HIV-2 reservoirs at levels comparable to those observed in untreated HIV-1 infection. Although the clinical spectrum is similar in the two infections, HIV-2 infection is associated with a much lower rate of CD4 T-cell decline and has a limited impact on the mortality of infected adults. Here we investigated HIV-2 infection of the human thymus, the primary organ for T-cell production. Human thymic tissue and suspensions of total or purified CD4 single-positive thymocytes were infected with HIV-2 or HIV-1 primary isolates using either CCR5 or CXCR4 coreceptors. We found that HIV-2 infected both thymic organ cultures and thymocyte suspensions, as attested to by the total HIV DNA and cell-associated viral mRNA levels. Nevertheless, thymocytes featured reduced levels of intracellular Gag viral protein, irrespective of HIV-2 coreceptor tropism and cell differentiation stage, in agreement with the low viral load in culture supernatants. Our data show that HIV-2 is able to infect the human thymus, but the HIV-2 replication cycle in thymocytes is impaired, providing a new model to identify therapeutic targets for viral replication control. IMPORTANCE HIV-1 infects the thymus, leading to a decrease in CD4 T-cell production that contributes to the characteristic CD4 T-cell loss.HIV-2 infection is associated with a very low rate of progression to AIDS and is therefore considered a unique naturally occurring model of attenuated HIV disease. HIV-2-infected individuals feature low to undetectable plasma viral loads, in spite of the numbers of circulating infected T cells being similar to those found in patients infected with HIV-1. We assessed, for the first time, the direct impact of HIV-2 infection on the human thymus. We show that HIV-2 is able to infect the thymus but that the HIV-2 replication cycle in thymocytes is impaired. We propose that this system will be important to devise immunotherapies that target viral production, aiding the design of future therapeutic strategies for HIV control.

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“…We also analyzed whether iron chelators have an effect on HIV-1 reverse transcription. We analyzed early RT by quantifying HIV-1 DNA for the early LTR (30). The established HIV-1 inhibitor AZT showed a statistically significant effect on HIV-1 LTR expression, whereas iron chelators showed no effect (Fig.…”

Section: Effects Of Ppy-based Iron Chelators On Hiv-1 Transcription Imentioning

confidence: 99%

Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

Kumari

Iordanskiy

Kovalskyy

et al. 2014

Antimicrob Agents Chemother

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dHIV-1 transcription is activated by the Tat protein, which recruits CDK9/cyclin T1 to the HIV-1 promoter. CDK9 is phosphorylated by CDK2, which facilitates formation of the high-molecular-weight positive transcription elongation factor b (P-TEFb) complex. We previously showed that chelation of intracellular iron inhibits CDK2 and CDK9 activities and suppresses HIV-1 transcription, but the mechanism of the inhibition was not understood. In the present study, we tested a set of novel iron chelators for the ability to inhibit HIV-1 transcription and elucidated their mechanism of action. Novel phenyl-1-pyridin-2yl-ethanone (

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Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors

Cited by 195 publications

References 52 publications

Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

Capsid-CPSF6 Interaction Is Dispensable for HIV-1 Replication in Primary Cells but Is Selected during Virus Passage In Vivo

Thymic HIV-2 Infection Uncovers Posttranscriptional Control of Viral Replication in Human Thymocytes

Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

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